1. Field of the Invention
This invention pertains to the field of in vitro enzymatic synthesis of gangliosides and related compounds.
2. Background
Gangliosides are a class of glycolipids, often found in cell membranes, that consist of three elements. One or more sialic acid residues are attached to an oligosaccharide or carbohydrate core moiety, which in turn is attached to a hydrophobic lipid (ceramide) structure which generally is embedded in the cell membrane. The ceramide moiety includes a long chain base (LCB) portion and a fatty acid (FA) portion. Gangliosides, as well as other glycolipids and their structures in general, are discussed in, for example, Lehninger, Biochemistry (Worth Publishers, 1981) pp. 287-295 and Devlin, Textbook of Biochemistry (Wiley-Liss, 1992). Gangliosides are classified according to the number of monosaccharides in the carbohydrate moiety, as well as the number and location of sialic acid groups present in the carbohydrate moiety. Monosialogangliosides are given the designation xe2x80x9cGMxe2x80x9d, disialogangliosides are designated xe2x80x9cGDxe2x80x9d, trisialogangliosides xe2x80x9cGTxe2x80x9d, and tetrasialogangliosides are designated xe2x80x9cGQxe2x80x9d. Gangliosides can be classified further depending on the position or positions of the sialic acid residue or residues bound. Further classification is based on the number of saccharides present in the oligosaccharide core, with the subscript xe2x80x9c1xe2x80x9d designating a ganglioside that has four saccharide residues (Gal-GalNAc-Gal-Glc-Ceramide), and the subscripts xe2x80x9c2xe2x80x9d, xe2x80x9c3xe2x80x9d and xe2x80x9c4xe2x80x9d representing trisaccharide (GalNAc-Gal-Glc-Ceramide), disaccharide (Gal-Glc-Ceramide) and monosaccharide (Gal-Ceramide) gangliosides, respectively.
Gangliosides are most abundant in the brain, particularly in nerve endings. They are believed to be present at receptor sites for neurotransmitters, including acetylcholine, and can also act as specific receptors for other biological macromolecules, including interferon, hormones, viruses, bacterial toxins, and the like. Gangliosides are have been used for treatment of nervous system disorders, including cerebral ischemic strokes. See, e g., Mahadnik et al. (1988) Drug Development Res. 15: 337-360; U.S. Pat. Nos. 4,710,490 and 4,347,244; Horowitz (1988) Adv. Exp. Med. and Biol. 174: 593-600; Karpiatz et al. (1984) Av. Exp. Med. and Biol. 174: 489-497.
Certain gangliosides are found on the surface of human hematopoietic cells (Hildebrand et al. (1972) Biochim. Biophys. Acta 260: 272-278; Macher et al. (1981) J. Biol. Chem. 256: 1968-1974; Dacremont et al. Biochim. Biophys. Acta 424: 315-322; Klock et al. (1981) Blood Cells 7: 247) which may play a role in the terminal granulocytic differentiation of these cells. Nojiri et al. (1988) J. Biol. Chem. 263: 7443-7446. These gangliosides, referred to as the xe2x80x9cneolactoxe2x80x9d series, have neutral core oligosaccharide structures having the formula [Galxcex2-(1,4)GlcNAcxcex2(1,3)]nGalxcex2(1,4)Glc, where n=1-4. Included among these neolacto series gangliosides are 3xe2x80x2-nLM1 (NeuAcxcex1(2,3)Galxcex2(1,4)GlcNAcxcex2(1,3)Galxcex2(1,4) Glcxcex2(1,1)-Ceramide) and 6xe2x80x2-nLM1 (NeuAcxcex1(2,6)Galxcex2(1,4)GlcNAcxcex2(1,3)Galxcex2(1,4)-Glcxcex2(1,1)-Ceramide).
The use of gangliosides as therapeutic reagents, as well as the study of ganglioside function, would be facilitated by convenient and efficient methods of synthesizing desired gangliosides. A combined enzymatic and chemical approach to synthesis of 3xe2x80x2-nLM1 and 6xe2x80x2-nLM1 has been described (Gaudino and Paulson (1994) J. Am. Chem. Soc. 116: 1149-1150). However, this and other previously available synthetic methods for ganglioside synthesis suffer from low efficiency and other drawbacks. Thus, a need exists for more efficient methods for synthesizing gangliosides. The present invention fulfills this and other needs.
The present invention provides methods for in vitro synthesis of glycosphingoids, including gangliosides, and other oligosaccharide-containing compounds. The methods involve the enzymatic transfer of carbohydrates, including sialic acids, to a sphingoid precursor. In particular, the methods involve contacting the sphingoid precursor with one or more glycosyltransferases and the corresponding sugar donor moiety for the glycosyltransferases, and other reactants required for glycosyltransferase activity, for a sufficient time and under appropriate conditions to transfer the sugar or sugars from the donor moiety to the sphingoid precursor. In some embodiments, one or more of the enzymatic reactions is carried out in the presence of an organic solvent, which increases the efficiency of the glycosylation reaction. The enzymatic step is typically preceded by hydrolysis of the fatty acid moiety from the ceramide; a fatty acid moiety can be reattached after completion of the glycosyltransferase reaction. As much as 100% efficiency is obtainable using the methods of the invention.
In one embodiment, the invention provides methods for adding one or more sialic acid residues to a glycosylated ceramide to form a ganglioside. The glycosylated ceramide is contacted with a sialyltransferase and a sialic acid donor moiety and other reactants required for sialyltransferase activity, under conditions such that sialic acid is transferred from the sialic acid donor moiety to the saccharide moiety of the glycosylated ceramide.